Reductase, gene thereof and method of using the same

ABSTRACT

The present invention relates to a protein which can asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to industrially advantageously produce an amide or ester compound of corresponding optically-active ortho-substituted mandelic acid, a DNA encoding the protein, a process for producing the protein from the DNA, and a process for asymmetrically reducing an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound.

This application is a 371 of PCT/JP2008/056258 filed Mar. 24, 2008,which claims priority to Japanese applications: 2007-074376, filed Mar.22, 2007 and 2008-011299, filed Jan. 22, 2008, respectively.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Apr. 13, 2010, is namedQ115164.txt and is 18,396 bytes in size.

TECHNICAL FIELD

The present invention relates to a novel reductase and a DNA encodingthe same, a recombinant vector containing the DNA, and a transformanttransformed with the recombinant vector. The present invention alsorelates to a process for producing an optically-active mandelic acidcompound which is an optically active alcohol, using the novel reductaseor the transformant.

BACKGROUND ART

Optically-active ortho-substituted mandelic acid compounds are compoundsuseful in production of medicines, agricultural chemicals and so on, andproduction processes using asymmetric reducing reagents or the like havebeen proposed, but in the process described in International PublicationNo. WO 0210101, further recrystallization or the like is performed aftera reaction in order to increase an optical purity and, also in areaction described in Organic Letters, 2005, vol. 7, No. 24, 5425-5427,it cannot be necessarily said that an optical yield of the reaction issufficient. In addition, has not been known a method of asymmetricallyreducing a keto group of a phenylglyoxylic acid compound, the grouphaving a substituent at an ortho-position and being in thesterically-complicated environment, to an optically-active mandelic acidcompound using a microorganism or the like.

DISCLOSURE OF THE INVENTION

The present invention provides a method of reducing a phenylglyoxalicacid compound having a substituent at an ortho-position to an opticallyactive mandelic acid compound with a good optical yield.

More specifically, the present invention provides a protein which canasymmetrically reduce an ortho-substituted phenylglyoxalic acid compoundto industrially advantageously produce an amide or ester compound ofcorresponding optically-active ortho-substituted mandelic acid, a DNAencoding the protein (hereinafter, abbreviated as invented DNA), atransformant comprising the DNA, a process for producing the proteinfrom the DNA, and a process for asymmetrically reducing anortho-substituted phenylglyoxalic acid compound to produce acorresponding optically-active ortho-substituted mandelic acid compound.

That is, the present invention provides:

1. A DNA comprising any nucleotide sequence of the following a) to d):

a) a nucleotide sequence encoding the amino acid sequence of SEQ IDNO:1;

b) a nucleotide sequence of a DNA: i) wherein the DNA has at least 90%sequence homology with a DNA comprising a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:1, and ii) wherein the nucleotidesequence encodes an amino acid sequence of a protein having the abilityto asymmetrically reduce an ortho-substituted phenylglyoxalic acidcompound to produce corresponding optically active ortho-substitutedmandelic acid;

c) a nucleotide sequence of a DNA: i) wherein the DNA hybridizes under astringent condition with a DNA comprising a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:1, and ii) wherein the nucleotidesequence encodes an amino acid sequence of a protein having the abilityto asymmetrically reduce an ortho-substituted phenylglyoxalic acidcompound to produce corresponding optically-active ortho-substitutedmandelic acid compound; and

d) the nucleotide sequence of SEQ ID NO:2;

2. A DNA in which a promoter functional in a host cell and the DNAaccording to the item 1 are operably linked;

3. A recombinant vector comprising the DNA according to the item 1 or 2;

4. A transformant in which the DNA of the item 2 or the recombinantvector of the item 3 has been introduced into a host cell;

5. The transformant according to the item 4, wherein the host cell is amicroorganism;

6. The transformant according to the item 4, wherein the host cell isEscherichia coli;

7. A transformant comprising the DNA of the item 1; 8. A process forproducing a transformant comprising a step of introducing therecombinant vector of the item 3 into a host cell;

9. A protein comprising any amino acid sequence of the following a) toe);

a) the amino acid sequence of SEQ ID NO:1;

b) an amino acid sequence: i) which is encoded by a nucleotide sequenceof a DNA having at least 90% sequence homology with a DNA comprising thenucleotide sequence of SEQ ID NO:2, and ii) which is an amino acidsequence of a protein having the ability to asymmetrically reduce anortho-substituted phenylglyoxalic acid compound to produce acorresponding optically-active ortho-substituted mandelic acid compound;

c) an amino acid sequence: i) which is encoded by a nucleotide sequenceof a DNA which hybridizes under a stringent condition with a DNAcomprising the nucleotide sequence of SEQ ID NO:2, and ii) which is anamino acid sequence of a protein having the ability to asymmetricallyreduce an ortho-substituted phenylglyoxalic acid compound to produce acorresponding optically-active ortho-substituted mandelic acid compound;

d) an amino acid sequence: i) in which one or a plurality of amino acidsare deleted, substituted or added in the amino acid sequence of SEQ IDNO:1, and ii) which is an amino acid sequence of a protein having theability to asymmetrically reduce an ortho-substituted phenylglyoxalicacid compound to produce a corresponding optically-activeortho-substituted mandelic acid compound; and

e) an amino acid sequence: i) which has at least 90% sequence homologywith the amino acid sequence of SEQ ID NO:1, and ii) which is an aminoacid sequence of a protein having the ability to asymmetrically reducean ortho-substituted phenylglyoxalic acid compound to produce acorresponding optically-active ortho-substituted mandelic acid compound;

10. A recombinant vector comprising the DNA of the item 1 and a DNAcomprising a nucleotide sequence encoding an amino acid sequence of aprotein having the ability to convert oxidized β-nicotineamide adeninedinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphateinto a reduced form;

11. The recombinant vector according to the item 10, wherein the proteinhaving the ability to convert oxidized β-nicotineamide adeninedinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphateinto a reduced form is glucose dehydrogenase;

12. The recombinant vector according to the item 11, wherein the proteinhaving glucose dehydrogenase activity is glucose dehydrogenase derivedfrom Bacillus megaterium;

13. A transformant, in which any of recombinant vector of the items 10to 12 has been introduced into a host cell;

14. The transformant according to the item 13, wherein the host cell isa microorganism;

15. The transformant according to the item 13, wherein the host cell isEscherichia coli;

16. A transformant comprising the DNA of the item 1 and a DNA comprisinga nucleotide sequence encoding an amino acid sequence of a proteinhaving the ability to convert oxidized β-nicotineamide adeninedinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphateinto a reduced form;

17. A process for producing an optically-active alcohol compoundcomprising reacting the protein of the item 9, or the transformant ofany of the items 4 to 7, and 13 to 16, or a treated product thereof witha prochiral carbonyl compound;

18. The process according to the item 17, wherein the prochiral carbonylcompound is an amide or ester compound of the ortho-substitutedphenylglyoxalic acid, and the corresponding optically-active alcoholcompound is an amide or ester compound of the optically-activeortho-substituted mandelic acid;

19. The process according to the item 18, wherein the amide or estercompound of the ortho-substituted phenylglyoxalic acid is a compoundrepresented by the formula (1):

(wherein R₁ represents an optionally substituted amino group, or anoptionally substituted alkoxy group, and R₂ represents an optionallysubstituted C1-8 alkyl group),and the amide or ester compound of the optically-activeortho-substituted active mandelic acid is an optically-active compoundrepresented by the formula (2):

(wherein R₁ and R₂ are as defined above, and a carbon atom with a symbolis an asymmetric carbon atom); and

20. Stenotrophomonas sp. SC-1 (FERM BP-10785); and the like.

According to the present invention, amide or ester compounds ofoptically-active ortho-substituted mandelic acid useful as medicines oragricultural chemicals, or intermediates thereof or the like can beproduced with a good optical purity.

BEST MODE FOR CARRYING OUT THE INVENTION

First, the invented DNA will be described.

The invented DNA can be obtained from a microorganism having the abilityto asymmetrically reduce an ortho-substituted phenylglyoxalic acidcompound to a corresponding optically-active ortho-substituted mandelicacid compound, for example, a microorganism belonging to genusStenotrophomonas such as Stenotrophomonas sp. SC-1 strain (FERM-BP10785). The invented DNA may be such a natural DNA, or a DNA produced byintroducing mutation into such a natural DNA by a method described later(site-directed mutagenesis, mutation treatment etc.).

The Stenotrophomonas sp. SC-1 strain has been deposited in InternationalPatent Organism Depositary (IPOD) National Institute of AdvancedIndustrial Science and Technology (AIST) Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, 305-8566 Japan, and the accession number ofFERM BP-10785 was assigned (date of original deposit: Feb. 21, 2007).Its mycological properties are as follows.

1. Colony Morphology (30° C., 24 Hours)

-   -   (1) Cellular morphology: rod-shaped, 0.7 to 0.8×1.5 to 2.0 μm    -   (2) Gram staining property: negative    -   (3) Presence or absence of spore: absence    -   (4) Presence or absence of mobility: presence

2. Colony Morphology on Nutrient Agar

-   -   Color of colony: pale yellow    -   Shape of colony: circular    -   Periphery of colony: entire periphery smooth    -   Prominence of colony: lens-like    -   Transparency: opaque

3. Physiological Properties

-   -   (1) Catalase: positive    -   (2) Oxidase: negative    -   (3) OF test: positive/negative

4. Nucleotide Sequence of DNA Encoding 16S Ribosomal RNA

About 500 bp of a nucleotide sequence of 16S ribosomal DNA was amplifiedby PCR from the Stenotrophomonas sp. SC-1 strain, and the nucleotidesequence was analyzed. Using the resulting nucleotide sequence of the16S ribosomal DNA, homology retrieval by BLAST was performed and, as aresult, the homology was 99.6%, showing highest homology with 16Sribosomal DNA of the Stenotrophomonas rhizophila standard strain. Alsoin BLAST retrieval using International Nucleotide Sequence Database,highest homology with 16S ribosomal DNA derived from genusStenotrophomonas was exhibited.

From the above mycological properties, the present bacterium wasidentified as Stenotrophomonas sp.

The “DNA which hybridizes under a stringent condition with a DNAcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO:1” in the invented DNA refers to a DNA such that, in Southernhybridization method described, for example, in “Cloning and Sequence”(supervised by Itaru Watanabe, edited by Masahiro Sugiura, 1989,published by Nouson Bunkasha), (1) it forms a DNA-DNA hybrid with a DNAcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO:1 by hybridization at 65° C. under a high ion concentration [forexample, 6×SSC (900 mM sodium chloride, 90 mM sodium citrate)], and (2)the hybrid is retained even after incubation at 65° C. for 30 minutesunder a low ion concentration [for example, 0.1×SSC (15 mM sodiumchloride, 1.5 mM sodium citrate)].

Specifically, examples include a DNA comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:1, a DNA comprising anucleotide sequence in which, in a nucleotide sequence encoding theamino acid sequence of SEQ ID No: 1, apart of nucleotides are deleted,substituted or added, a DNA having at least 90% sequence homology,preferably at least 95% sequence homology, more preferably at least 99%sequence homology with a DNA comprising a nucleotide sequence encodingthe amino acid sequence of SEQ ID No: 1, and the like.

The DNA may be a DNA cloned from naturally occurring DNAs, a DNA inwhich, in a nucleotide sequence of this cloned DNA, deletion,substitution or addition of a part of nucleotides is artificiallyintroduced, or an artificially synthesized DNA. The sequence homologycan be calculated using a tool for sequence analysis such as BESTFITprogram supplied by UWGCG Package (Devereux et al (1984) Nucleic AcidsResearch 12, p 387-395), and PILEUP and BLAST algorism (Altschul S. F.(1993) J Mol Evol 36:290-300; Altschul S. F. (1990) Mol Biol215:403-10).

The invented DNA can be prepared, for example, as follows.

The invented DNA can be prepared by amplifying a DNA comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, aDNA comprising a nucleotide sequence encoding an amino acid sequence inwhich one or a plurality of amino acids are deleted, substituted oradded in the amino acid sequence of SEQ ID NO:1 and/or a DNA comprisingthe nucleotide sequence of SEQ ID NO:2, by preparing a DNA library froma microorganism belonging to genus Stenotrophomonas such asStenotrophomonas sp. according to conventional genetic engineeringprocedures (e.g. method described in “New Cellular TechnologyExperimental Protocol” (edited by the Institute of Medical Science, theUniversity of Tokyo, Anticancer Research Section, Shujunsha, 1993)), andperforming PCR employing the prepared DNA library as a template, andusing appropriate primers.

In addition, the invented DNA can be prepared by amplifying a DNAcomprising the nucleotide sequence of SEQ ID NO: 2 by performing PCRemploying the DNA library as a template, and using an oligonucleotidecomprising the nucleotide sequence of SEQ ID NO:12 and anoligonucleotide comprising the nucleotide sequence of SEQ ID NO:13 asprimers.

Examples of the condition of the PCR include the condition of heating areaction solution obtained by mixing each 20 μM of four kinds of dNTPs,each 15 pmol of two kinds of oligonucleotide primers, 1.3 U ofTaqpolymerase, and a DNA library as a template at 94° C. for 2 minutes,thereafter, performing 10 times of a cycle of 94° C. (10 seconds)-65° C.(30 seconds)-72° C. (90 seconds), then, 20 times of a cycle of 94° C.(10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), andfurther retaining the solution at 72° C. for 7 minutes.

In addition, a restriction enzyme recognition sequence or the like maybe added to the 5′ end and/or the 3′ end of the primer used in the PCR.

In addition, also by performing PCR employing the DNA library as atemplate, and using an oligonucleotide comprising a partial nucleotidesequence selected from nucleotide sequences encoding the amino acidsequence of SEQ ID NO:1 (e.g. an oligonucleotide comprising a nucleotidesequence of about 14 or more nucleotides on the 5′-terminal sideencoding the amino acid sequence of SEQ ID No:1) or the like and anoligonucleotide of about 14 or more nucleotides comprising a nucleotidesequence complementary to a nucleotide sequence adjacent to aDNA-insertion site of a vector used in construction of the DNA libraryas primers, a DNA comprising a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:1, a DNA comprising a nucleotide sequenceencoding an amino acid sequence in which one or a plurality of aminoacids are deleted, substituted or added in the amino acid sequence ofSEQ ID NO:1, or the like can be amplified to prepare the invented DNA.

The thus amplified DNA is cloned into a vector according to a methoddescribed in “Molecular Cloning: A Laboratory Manual 2^(nd) edition”(1989), Cold Spring Harbor Laboratory Press, “Current Protocols inMolecular Biology” (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X orthe like, whereby the invented vector can be obtained. Specific examplesof the vector used include pUC119 (manufactured by Takara Shuzou),pTV118N (manufactured by Takara Shuzou), pBluescriptII (manufactured byToyobo), pCR2.1-TOPO (manufactured by Invitrogen), pTrc99A (manufacturedby Pharmacia), pKK223-3 (manufactured by Pharmacia) and the like.

In addition, the invented DNA can also be obtained, for example, byhybridizing, as a probe, a DNA comprising a nucleotide sequence of about15 or more nucleotides having a partial nucleotide sequence selectedfrom nucleotide sequences encoding the amino acid sequence of SEQ IDNO:1 to a DNA library inserted in a vector derived from a microorganismor a phage under the condition described later, and detecting a DNA towhich the probe specifically binds.

Examples of a method of hybridizing a probe to a chromosomal DNA or aDNA library include colony hybridization and plaque hybridization, andthe method can be selected depending on a kind of a vector used inpreparing a library.

When the library used is prepared using a plasmid vector, colonyhybridization may be utilized. Specifically, a transformant is obtainedby introducing a DNA of a library into a host microorganism, the resulttransformant is diluted, and then the dilution is seeded on an agarmedium, and this is cultured until a colony appears.

When the library used is prepared using a phage vector, plaquehybridization may be utilized. Specifically, a host microorganism and aphage of a library are mixed under the infectable condition, the mixtureis further mixed with a soft agar medium, and then the mixture is seededon an agar medium, and this is cultured until a plaque appears.

Then, in either hybridization, a membrane is placed on an agar medium onwhich the culturing has been performed, and a transformant or a phage isadsorbed or transferred on the membrane. This membrane isalkali-treated, and subjected to neutralization treatment and, then, aDNA is subjected to fixation on the membrane. More specifically, in thecase of plaque hybridization, a nitrocellulose membrane or a nylonmembrane (e.g. Hybond-N⁺ (trade mark, Amersham)) is placed on the agarmedium, and this is allowed to stand still for about 1 minute to adsorbor transfer phage particles onto the membrane. Then, the membrane isimmersed in an alkali solution (e.g. 1.5 M sodium chloride, 0.5 M sodiumhydroxide) for about 3 minutes to lyse the phage particles, whereby aphage DNA is eluted on the membrane, and this is immersed in aneutralization solution (e.g. 1.5 M sodium chloride, 0.5 MTris-hydrochloric acid buffer pH 7.5) for about 5 minutes. Then, themembrane is washed with a washing solution (e.g. 0.3 M sodium chloride,30 mM citric acid, 0.2 M Tris-hydrochloric acid buffer pH 7.5) for about5 minutes, thereafter, the phage DNA is fixed on the membrane, forexample, by heating at about 80° C. for about 90 minutes.

The thus prepared membrane is used to perform hybridization using theDNA as a probe. Hybridization can be performed, for example, accordingto the description of J. Sambrook, E. F. Frisch, T. Maniatis “MolecularCloning: A Laboratory Manual 2^(nd) edition (1989)” Cold Spring HarborLaboratory Press or the like.

The DNA used in the probe may be labeled with a radioactive isotopeelement, or may be labeled with a fluorescent dye.

Examples of a method of labeling the DNA used in the probe with aradioactive isotope element include a method of performing PCR using theDNA used in the probe as a template, by replacing dCTP in a PCR reactionsolution with (α-³²P) dCTP utilizing, for example, Random PrimerLabeling Kit (manufactured by Takara Shuzou).

In addition, when the DNA used in the probe is labeled with afluorescent dye, for example, ECL Direct Nucleic Acid Labeling andDetection System manufactured by Amersham can be used.

Hybridization can be performed, for example, as follows.

A pre-hybridization solution containing 450 to 900 mM sodium chloride,45 to 90 mM sodium citrate, sodium dodecylsulfate (SDS) at aconcentration of 0.1 to 1.0% by weight, a denatured non-specific DNA ata concentration of 0 to 200 μl/ml, and optionally containing albumin,Ficoll, polyvinylpyrrolidone or the like at a concentration of 0 to 0.2%by weight, respectively (preferably, a pre-hybridization solutioncontaining 900 mM sodium chloride, 90 mM sodium citrate, 1.0% by weightof SDS and 100 μl/ml denatured Calf-thymus DNA) is prepared at a ratioof 50 to 200 μl per 1 cm² of the membrane prepared above, and themembrane is immersed in the pre-hybridized solution, and retained at 42to 65° C. for 1 to 4 hours.

Then, for example, a solution obtained by mixing a hybridizationsolution containing 450 to 900 mM sodium chloride, 45 to 90 mM sodiumcitrate, SDS at a concentration of 0.1 to 1.0% by weight, a denaturednon-specific DNA at a concentration of 0 to 200 μg/ml, and optionallycontaining albumin, Ficoll, polyvinylpyrrolidone or the like at aconcentration of 0 to 0.2% by weight, respectively (preferably, ahybridization solution containing 900 mM sodium chloride, 90 mM sodiumcitrate, 1.0% by weight of SDS and 100 μg/ml denatured Calf-thymus DNA),and the probe prepared by the aforementioned method (1.0×10⁴ to 2.0×10⁶cpm equivalent amount per 1 cm² of the membrane) is prepared at a ratioof 50 to 200 μl per 1 cm² of the membrane, and the membrane is immersedin the hybridization solution, and retained at 42 to 65° C. for 12 to 20hours.

After the hybridization, the membrane is removed, and washed using awashing solution containing 15 to 300 mM sodium chloride, 1.5 to 30 mMsodium citrate and 0.1 to 1.0% by weight of SDS at 42 to 65° C.(preferably, a washing solution containing 15 mM sodium chloride, 1.5 mMsodium citrate and 1.0% by weight of SDS at 65° C.) or the like. Thewashed membrane is mildly rinsed with 2×SSC (300 mM sodium chloride, 30mM sodium citrate), and dried. This membrane is subjected to, forexample, autoradiography, and a position of the probe on the membrane isdetected, whereby a clone corresponding to a position on the membrane ofa DNA which hybridizes with the probe used is specified on the originalagar medium, and this is picked up, whereby a clone having the DNA isisolated.

From a cultured bacterial cell obtained by culturing the thus obtainedclone, the invented DNA can be prepared.

The DNA prepared as described above is cloned into a vector according toa method described in “Molecular Cloning: A Laboratory Manual 2^(nd)edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocolsin Molecular Biology” (1987), John Wiley & Sons, Inc. ISBNO-471-50338-Xor the like, whereby the invented recombinant vector can be obtained.Examples of the vector used include, pUC119 (manufactured by TakaraShuzou), pTV118N (manufactured by Takara Shuzou), pBluescriptII(manufactured by Toyobo), pCR2.1-TOPO (manufactured by Invitrogen),pTrc99A (manufactured by Pharmacia), pKK223-3 (manufactured byPharmacia) and the like.

In addition, a nucleotide sequence of the DNA can be analyzed by adideoxy terminator method described in F. Sanger, S. Nicklen, A. R.Coulson, Proceeding of Natural Academy of Science U.S.A. (1977) 74:5463-5467, or the like. For preparing a sample for nucleotide sequenceanalysis, a commercially available reagent such as ABI PRISM DyeTerminator Cycle Sequencing Ready Reaction Kit of Perkin Elmer may beused.

Confirmation that the DNA obtained as described above encodes an aminoacid sequence of a protein having the ability to asymmetrically reducean ortho-substituted phenylglyoxalic acid compound, typically, an amideor ester compound thereof (e.g.2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide) to acorresponding optically-active ortho-substituted mandelic acid compound,typically, an amide or ester compound thereof (e.g.(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide)can be performed, for example, as follows.

First, the DNA obtained as described above is inserted into a vector sothat it can be linked to the downstream of a promoter functional in ahost cell as described later, and this vector is introduced into a hostcell to obtain a transformant. Then, a culture of the transformant ismade to act on an ortho-substituted phenylglyoxalic acid compound (e.g.an amide or ester compound of that compound, specifically, for example,2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide). Byanalyzing an amount of a corresponding optically-activeortho-substituted mandelic acid compound (e.g. an amide or estercompound thereof, specifically, for example,(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide)in the reaction product, it can be confirmed that the resulting DNAencodes an amino acid sequence of a protein having such ability.

In order to express the invented DNA in a host cell, for example, a DNAin which a promoter functional in a host cell and the invented DNA areoperably linked is introduced into a host cell.

Herein, the “operably” means that upon transformation of a host cell byintroduction of the DNA into a host cell, the invented DNA is in thestate where it is bound to a promoter so as to be expressed undercontrol of the promoter. Examples of the promoter include a promoter ofthe lactose operon of Escherichia coli, a promoter of the tryptophanoperon of Escherichia coli, and a synthetic promoter functional inEscherichia coli such as a tac promoter and a trc promoter, and apromoter which controls expression of the invented DNA inStenotrophomonaes sp. may be utilized.

Generally, a recombinant vector obtained by inserting a DNA operablylinked to a promoter functional in a host cell into the aforementionedvector is introduced into a host cell. When a vector including aselection marker gene (e.g. an antibiotic resistance imparting gene suchas a kanamycin resistant gene, a neomycin resistant gene etc.) is usedas a vector, a transformant into which the vector is introduced can beselected using a phenotype of the selection marker gene or the like asan index.

Examples of the host cell into which the invented DNA operably linked toa promoter functional in a host cell, or the invented recombinant vectoris introduced include a microorganism belonging to genus Escherichia,genus Bacillus, genus Corynebacterium, genus Staphylococcus, genusStreptomyces, genus Saccharomyces, genus Kluyveromyces, genus Pichia,genus Rhodococcus and genus Aspergillus.

A method of introducing the invented DNA operably linked to a promoterfunctional in a host cell, or the invented recombinant vector into ahost cell may be a conventionally used introduction method depending ona host cell used, and examples include a calcium chloride methoddescribed in “Molecular Cloning: A Laboratory Manual 2^(nd) edition”(1989), Cold Spring Harbor Laboratory Press, “Current Protocols inMolecular Biology” (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X orthe like, and an electroporation method described in “Methods inElectroporation: Gene Pulser/E. coli Pulser System” Bio-RadLaboratories, (1993) or the like.

In order to select a transformant into which the invented DNA operablylinked to a promoter functional in a host cell, or the inventedrecombinant vector has been introduced, for example, the transformantmay be selected using, as an index, a phenotype of a selection markergene contained in the vector.

Confirmation that the transformant harbors the invented DNA can beperformed by confirmation of a restriction enzyme site, analysis of anucleotide sequence, Southern hybridization, Western hybridization orthe like according to conventional methods described in “MolecularCloning: A Laboratory Manual 2^(nd) edition” (1989), Cold Spring HarborLaboratory Press or the like.

Then, the invented protein will be described.

As the “amino acid in which one or a plurality of amino acids aredeleted, substituted or added in the amino acid sequence of SEQ IDNO:1”, an amino acid sequence in which one amino acid is deleted,substituted or added in the amino acid sequence of SEQ ID NO:1 ispreferable.

The invented protein can be produced, for example, by culturing atransformant comprising the invented DNA.

As a medium for culturing the transformant, for example, various mediaappropriately containing a carbon source or a nitrogen source, anorganic salt or an inorganic salt, or the like which are normally usedin culturing a host cell such as a microorganism can be used.

Examples of the carbon source include sugars such as glucose, dextrinand sucrose, sugar alcohols such as glycerol, organic acids such asfumaric acid, citric acid and pyruvic acid, animal oils, vegetable oilsand beewax. The amount of these carbon sources to be added to a mediumis usually around 0.1 to 30% (w/v) relative to the culturing solution.

Examples of the nitrogen source include natural organic nitrogen sourcessuch as meat extract, peptone, yeast extract, malt extract, soybeanpowder, corn steep liquor, cottonseed powder, dry yeast and casaminoacid, amino acids, ammonium salts of inorganic acids such as sodiumnitrate, ammonium salts of inorganic acids such as ammonium chloride,ammonium sulfate and ammonium phosphate, ammonium salts of organic acidssuch as ammonium fumarate and ammonium citrate, and urea. Among them,ammonium salts of organic acids, natural organic nitrogen sources, aminoacids and the like can also be used as a carbon source in many cases.The amount of these nitrogen sources to be added to a medium is usuallyaround 0.1 to 30% (w/v) relative to the culturing solution.

Examples of the organic salts or the inorganic salts include chlorides,sulfates, acetates, carbonates and phosphates of potassium, sodium,magnesium, iron, manganese, cobalt, zinc and the like. Specific examplesthereof include sodium chloride, potassium chloride, magnesium sulfate,ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate,copper sulfate, sodium acatate, potassium carbonate, monopotassiumhydrogen phosphate and dipotassium hydrogen phosphate. The amount ofthese organic salts and/or inorganic salts to be added to a medium isusually around 0.0001 to 5% (w/v).

Further, in the case of a transformant into which a gene in which a typeof a promoter induced by allolactose such as a tac promoter, a trcpromoter and a lac promoter, and the invented DNA are linked operably,as an induction agent for inducing production of the invented protein,for example, isopropyl thio-β-D-galactoside (IPTG) may be added to amedium at a small amount.

Culturing of a transformant comprising the invented DNA can be performedaccording to a method which is conventionally used in culturing a hostcell such as a microorganism, and examples thereof include liquidculturing and solid culturing such as tube shaking culturing,reciprocating shaking culturing, jar fermenter culturing, tank culturingand the like.

A culturing temperature can be appropriately changed in such a rangethat the transformant can be grown, and is usually about 15 to 40° C. ApH of a medium is preferably in a range of about 6 to 8. A culturingtime is different depending on the culturing condition and, usually,about 1 day to about 5 days is preferable.

As a method of purifying the invented protein from a culture of thetransformant harboring the invented DNA, a method which is normally usedin purification of a protein can be applied, and, for example, thefollowing methods can be exemplified.

First, after cells are collected from a culture of the transformant bycentrifugation or the like, these cells are ground by a physicalgrinding method such as ultrasonic treatment, dino mill treatment andFrench press treatment, or a chemical grinding method using surfactantsor a bacteriolytic enzyme such as lysozyme. A cell-free extract isprepared by removing impurities from the resulting grinding solution bycentrifugation, membrane filter filtration or the like, and this isfractionated by appropriately using a separation purification methodsuch as cationic exchange chromatography, anion exchange chromatography,hydrophobic chromatography, gel filtration chromatography and metalchelate chromatography, whereby the invented protein can be purified.

Examples of a carrier used in chromatography include an insolublepolymer carrier such as cellulose, dextrin and agarose into which acarboxymethyl (CM) group, a diethylaminoethyl (DEAE) group, a phenylgroup or a butyl group is introduced. A commercially availablecarrier-filled column may be used, and examples of the commerciallyavailable carrier-filled column include Q-Sepharose FF, Phenyl-SepharoseHP (trade name, both manufactured by Amersham Pharmacia Biotech),TSK-gel G3000SW (trade name, manufactured by Tosoh) and the like.

In order to select a fraction containing the invented protein, forexample,2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide isasymmetrically reduced, and the fraction may be selected using, as anindex, the ability to preferentially produce(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.

The transformant of the present invention, and an treated productthereof can be used for asymmetrically reducing the ortho-substitutedphenylglyoxalic acid compound represented by the formula (1) (e.g. anester or amide compound thereof) which is a prochiral carbonyl compoundto produce an optically active ortho-substituted mandelic compoundrepresented by the formula (2) (e.g. an amide or ester compound thereof)which is an optically active alcohol compound.

In the ortho-substituted phenylglyoxalic acid compound of the formula(1), and the ortho-substituted mandelic acid compound of the formula(2), a substituent represented by R₁ will be described below.

Examples of an optionally substituted amino group represented by R₁include, in addition to an amino group, a C1-6 alkylamino group such asa methlyamino group, an ethylamino group, a propylamino group, anisopropylamino group, a butylamino group, an isobutylamino group, at-butylamino group, a pentylamino group, and a hexylamino group. Inaddition, examples of an optionally substituted alkoxy group include aC1-8 alkoxy group such as a methoxy group, an ethoxy group, a propoxygroup, an isopropoxy group, a butoxy group, a pentyloxy group, ahexyloxy group, a heptyloxy group and an octyloxy group.

Then, a substituent represented by R₂ will be described below.

Examples of a C1-8 alkyl group in an optionally substituted C1-8 alkylgroup represented by R₂ include a methyl group, an ethyl group, a propylgroup, an isopropyl group, a butyl group, a pentyl group, a hexyl group,a heptyl group and an octyl group.

Examples of a substituent of such a C1-8 alkyl group include anoptionally substituted alkoxy group and an optionally substitutedaryloxy group.

Examples of an optionally substituted alkoxyalkyl group represented byR₂ include a C1-8 alkyl group substituted with a C1-4 alkoxy group suchas a methoxymethyl group, a methoxyethyl group, a methoxypropyl group, amethoxybutyl group, a methoxypentyl group, a methoxyhexyl group, amethoxyheptyl group, a methoxyoctyl group, an ethoxymethyl group, anethoxyethyl group, an ethoxypropyl group, an ethoxybutyl group, anethoxypentyl group, an ethoxyhexyl group, an ethoxyheptyl group, anethoxyoctyl group, a propoxymethyl group, a propoxyethyl group, apropoxypropyl group, a propoxybutyl group, a propoxypentyl group, apropoxyhexyl group, a propoxyheptyl group, a propoxyoctyl group, abutoxymethyl group, a butoxyethyl group, a butoxypropyl group, abutoxybutyl group, a butoxypentyl group, a butoxyhexyl group, abutoxyheptyl group and a butoxyoctyl group.

Examples of an optionally substituted aryloxy group include an aryloxygroup represented by the formula (3):

(wherein A, B and C are the same or different from one another, andrepresent a hydrogen atom, an alkyl group, an alkenyl group, an alkynylgroup, a cycloakly group, a cycloalkenyl group, an alkoxy group, ahalogenated alkyl group, a hydroxyalkyl group, an alkoxyalkyl group, anaminoalkyl group, an alkylaminoalkyl group, a halogen atom, a nitrogroup, a cyano group, an aralkyl group optionally substituted with analkoxy group, an aryl group optionally substituted with a substituentselected from a halogen atom and an alkoxy group, an alkylthio group, anamino group, or an alkylamino group).

The substituent represented by A, B or C in the formula (3) will bedescribed below.

Examples of the alkyl group include a C1-4 alkyl group such as a methylgroup, an ethyl group, a propyl group, and a butyl group.

Examples of the alkenyl group include a C2-4 alkenyl group such as avinyl group, an allyl group, and a crotyl group.

Examples of the alkynyl group include a C2-4 alkynyl group such as anethynyl group, a propargyl group, and a butynyl group.

Examples of the cycloalkyl group include a C3-6 cycloalkyl group such asa cyclopropyl group, a cyclopentyl group, and a cyclohexyl group.

Examples of the cycloalkenyl group include a C5-6 cycloalkenyl groupsuch as a cyclopentenyl group, and a cyclohexenyl group.

Examples of As the alkoxy group include a C1-4 alkoxy group such as amethoxy group, an ethoxy group, a propoxy group, and a butoxy group.

Examples of the halogenated alkyl group include a C1-3 haloalkyl groupsuch as a trifluoromethyl group, a trichloromethyl group, adifluoromethyl group, a chloromethyl group, a 2-bromoethyl group, and a1,2-dichloropropyl group.

Examples of the hydroxyalkyl group include a C1-2 alkyl groupsubstituted with hydroxyl such as a hydroxymethyl group, and a 1-,2-hydroxyethyl group.

Examples of the alkoxyalkyl group include a C1-2 alkyl group substitutedwith C1-2 alkoxy such as a methoxymethyl group, a methoxyethyl group, anethoxymethyl group, and an ethoxyethyl group.

Examples of the aminoalkyl group include a C1-2 alkyl group substitutedwith monoamino, or a C1-2 alkyl group substituted with diamino, such asan aminomethyl group, and a 1-, 2-aminoethyl group.

Examples of the alkylaminoalkyl group include a C1-2 alkyl groupsubstituted with (C1-2)alkylamino, or a C1-2 alkyl group substitutedwith di(C1-2)alkylamino, such as a methylaminomethyl group, adimethylaminomethyl group, and a diethylaminomethyl group.

Examples of the halogen atom include a fluorine atom, a chlorine atom, abromine atom, or an iodine atom.

Examples of the aralkyl group optionally substituted with an alkoxygroup include a C7-8 aralkyl group optionally substituted with a C1-2alkoxy group, such as a benzyl group, a phenethyl group, and a4-methoxybenzyl group.

Examples of the aryl group optionally substituted with a group selectedfrom a halogen atom and an alkoxy group include a C6-10 aryl groupoptionally substituted with a group selected from a halogen atom(fluorine atom, chlorine atom, bromine atom and iodine atom) and a C1-2alkoxy group such as a 2-, 3-, 4-chlorophenyl group, a 2-, 3-,4-methylphenyl group, a 2-, 3-, 4-methoxyphenyl group, a phenyl group, a1-naphthyl group, and a 2-naphthyl group.

Examples of the alkylthio group include a C1-3 alkylthio group such as amethylthio group, an ethylthio group and a propylthio group.

Examples of the alkylamino group include a C1-6 alkylamino group such asa methylamino group, an ethylamino group, a propylamino group, anisopropylamino group, a butylamino group, an isobutylamino group, at-butylamino group, a pentylamino group, and a hexylamino group.

From an ortho-substituted phenylglyoxalic acid compound having theoptionally substituted aryloxy group and being represented by theformula (1′):

(wherein R₁ represents an optionally substituted amino group, or anoptionally substituted alkoxy group, and A, B and C are the same ordifferent from one another, and represent a hydrogen atom, an alkylgroup, an alkenyl group, an alkynyl group, a cycloalkyl group, acycloaklenyl group, an alkoxy group, a halogenated alkyl group, ahydroxyalkyl group, an alkoxyalkyl group, an aminoalkyl group, analkylaminoalkyl group, a halogen atom, a nitro group, a cyano group, anaralkyl group optionally substituted with an alkoxy group, an aryl groupoptionally substituted with a substituent selected from a halogen atomand an alkoxy group, an alkylthio group, an amino group or an alkylaminogroup), a compound represented by the formula (2′):

(wherein R₁, A, B and B are as defined above, and a hydrogen atom with asymbol is an asymmetric carbon atom) is obtained.

In the compounds of the formula (1) and (1′), as the optionallysubstituted amino group represented by R₁, a methylamino group ispreferable and, as the optionally substituted alkoxy group, for example,a methoxy group or an ethoxy group is preferable. In addition, as theoptionally substituted C1-8 alkyl group represented by R₂, a methylgroup is preferable, as the optionally substituted alkoxyalkyl group,for example, a methoxymethyl group is preferable, as the optionallysubstituted aryloxy alkyl group, for example, a dimethylphenoxymethylgroup is preferable and, more particularly,2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide, ethyl2-(2-methyl-phenyl)oxoacetate,2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide, ethyl2-(2-methoxymethyl-phenyl)-2-oxoacetate,2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, ormethyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-oxoacetate isexemplified.

In the method of the present invention, when such an ortho-substitutedphenylglyoxalic acid compound is a substrate, the compound of theformula (2) or the formula (2′) is obtained, and as a specific compound,an optically-active substance of2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide, ethyl2-(2-methyl-phenyl)-2-hydroxy-acetate,2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide, ethyl2-(2-methoxymethyl-phenyl)-2-hydroxyacetate,2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide,or methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-hydroxyacetate isobtained, respectively.

The method is usually performed in the presence of water, and a coenzymesuch as reduced nicotineamide adenine dinucleotide phosphate(hereinafter, referred to as NADPH). The water used thereupon may be anaqueous buffer solution. Examples of a buffer used in the aqueous buffersolution include an alkali metal salt of phosphoric acid such as sodiumphosphate and potassium phosphate, an aqueous sodium acetate solution,an alkali metal salt of acetic acid such as potassium acetate, and amixture thereof.

In the method, in addition to water, an organic solvent may be presenttogether. Examples of the organic solvent which may be present togetherinclude ethers such as t-butyl methyl ether, diisopropyl ether andtetrahydrofuran, esters such as ethyl formate, ethyl acetate, propylacetate, butyl acetate, ethyl propionate and butyl propionate,hydrocarbons such as toluene, hexane, cyclohexane, heptane andisooctane, alcohols such as methanol, ethanol, 2-propanol, butanol andt-butyl alcohol, organic sulfur compounds such as dimethyl sulfoxide,ketones such as acetone, nitriles such as acetonitrile, and a mixturethereof.

For example, a reaction in the method is performed by mixing throughstirring, shaking or the like in the state where water, an amide orester compound of ortho-substituted phenylglyoxalic acid (e.g.2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide), andNADPH, together with the invented protein, or a transformant producingthe same, or a treated product thereof and, if necessary, further anorganic solvent or the like are contained.

A pH at a reaction in the method can be appropriately selected, and isusually in a range of a pH of 3 to 10. In addition, a reactiontemperature can be appropriately selected, and is usually in a range of0 to 60° C. from the viewpoint of stability of a raw material and aproduct, and a reaction rate.

An endpoint of the reaction can be determined, for example, by tracingthe amount of an amide or ester compound of ortho-substitutedphenylglyoxalic acid (e.g.2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide) in thereaction solution by liquid chromatography or the like.

A reaction time can be appropriately selected, and is usually in a rangeof 0.5 hour to 10 days.

The recovery of an amide or ester compound of ortho-substitutedoptically active mandelic acid (e.g.(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide)from the reaction solution may be performed by a generally knownarbitrary method.

Examples thereof include a purification method by performingpost-treatment such as organic solvent extraction operation andconcentration operation of the reaction solution, if necessary, incombination with column chromatography, distillation or the like.

The invented protein, a transformant producing the same, or a treatedproduct thereof can be used in the method in various forms.

Examples of the specific form include a culture of a transformantcomprising the invented DNA, and examples of the treated product of thetransformant include a cell-free extract, a crude purified protein, apurified protein and the like, and an immobilized product thereof.Herein, examples of the treated product of the transformant include alyophilized transformant, an organic solvent-treated transformant, a drytransformant, a transformant ground product, a self-digestion product ofa transformant, an ultrasonic-treated product of a transformant, atransformant extract, and an alkali-treated product of a transformant.In addition, examples of a method of obtaining an immobilized productinclude a carrier binding method (a method of adsorbing the inventedprotein or the like onto an inorganic carrier such as silica gel and aceramic, cellulose and an ion-exchanged resin), and an encapsulatingmethod (a method of confining the invented protein or the like in anetwork structure of a polymer such as polyacrylamide, sulfur-containingpolysaccharide gel (e.g. carrageenan gel), alginic acid gel and agargel).

In addition, in view of industrial production using the transformantharboring the invented DNA, a method using a treated product obtained bykilling the transformant is preferable rather than a method using aliving transformant in that the restriction of a production facility issmall. Examples of a method of kill treatment therefor include aphysical sterilization method (heating, drying, freezing, light ray,ultrasound, filtration, powder distribution), and a sterilization methodusing a chemical (alkali, acid, halogen, oxidizing agent, sulfur, boron,arsenic, metal, alcohol, phenol, amine, sulfide, ether, aldehyde,ketone, cyan and antibiotic). Generally, it is desirable to select atreating method which inactivates enzyme activity of the inventedprotein as little as possible, and has little influence such asremaining in a reaction system, and contamination, among thesesterilization methods.

In addition, a process for producing an amide or eater compound of theoptically-active ortho-substituted mandelic acid of the presentinvention is performed in the presence of a coenzyme such as NADPH, inan amide or ester compound of the ortho-substituted phenylglyoxalic acid(e.g. 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide),as an asymmetrical reducing reaction proceeds, the NADPH is convertedinto oxidized β-nicotineamide adenine dinucleotide phosphate(hereinafter, referred to as NADP⁺). Since the NADP⁺ generated byconversion can be returned to original NADPH by a protein having theability to convert NADP⁺ into reduced form (NADP), a protein having theability to convert NADP⁺ into NADPH can be present together in thereaction system of the method.

Examples of the protein having the ability to convert oxidizedβ-nicotineamide adenine dinucleotide (hereinafter, referred to as NAD⁺),or NADP⁺ into reduced β-nicotineamide adenine dinucleotide (hereinafter,referred to as NADH), or NADPH include glucose dehydrogenase, alcoholdehydrogenase, aldehyde dehydrogenase, amino acid dehydrogenase, organicdehydrogenase (malate dehydrogenase etc.) and the like.

In addition, when the protein having the ability to convert NAD⁺, orNADP⁺ into NADH, or NADPH is glucose dehydrogenase, by allowing glucoseand the like to be present together in a reaction system, activity ofthe protein is enhanced in some cases, and these may be added, forexample, to the reaction solution.

In addition, the protein may be an enzyme itself, or may be presenttogether in the form of a microorganism having the enzyme, or a treatedproduct of the microorganism in the reaction system. Further, it may bea transformant containing a gene having a nucleotide sequence encodingan amino acid sequence of a protein having the ability to convert NAD⁺,or NADP⁺ into NADH, or NADPH, or a treated product thereof. Herein, thetreated product means an equivalent of the aforementioned “treatedproduct of a transformant”.

Further, in the process for producing an amide or ester compound of theortho-substituted optically-active mandelic acid of the presentinvention, the process may also be performed using a transformantsimultaneously comprising a DNA comprising a nucleotide sequenceencoding an amino acid sequence of a protein having the ability toconvert NAD⁺, or NADP⁺ into NADH, or NADPH such as glucosedehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, amino aciddehydrogenase, and organic dehydrogenase (malate dehydrogenase etc.).

In this transformant, examples of a method of introducing both DNAs intoa host cell include a method of introducing a single vector containingboth DNAs into a host cell, a method of transforming a host cell with arecombinant vector in which both DNAs are separately introduced into aplurality of vectors having different replication origins, and the like.Further, one DNA or both DNAs may be introduced into a chromosome of ahost cell.

In addition, as the method of introducing a single vector containingboth DNAs into a host cell, for example, regions involved in expressioncontrol such as a promoter and a terminator may be connected to bothDNAs to construct a recombinant vector, or a recombinant vectorexpressing a gene as an operon containing a plurality of cistrons suchas lactose operon may be constructed.

EXAMPLES

Hereinafter, the present invention will be described in more detail byway of Examples or the like, but the present invention is not limited bythose Examples at all.

Reference Example 1 Preparation of Chromosomal DNA

Each 100 ml of a medium (obtained by dissolving 2 g of glucose, 0.5 g ofpolypeptone, 0.3 g of yeast extract, 0.3 g of meat extract, 0.2 g ofammonium sulfate, 0.1 g of potassium dihydrogen phosphate, and 0.05 g ofmagnesium sulfate heptahydrate, and adjusting a pH to 6 with 2 N HCl)was placed into two 500-ml flasks, and was sterilized at 121° C. for 15minutes. To this was added each 0.3 ml of a culture of Stenotrophomonassp. SC-1 strain (FERM-BP 10785) cultured (30° C., 48 hours, shakingculturing) in a medium of the same composition, followed by shakingculturing at 30° C. for 24 hours. Thereafter, the resulting culture wascentrifuged (8000 rpm, 4° C., 10 minutes), and the resulting precipitatewas collected. The precipitate was washed with 50 ml of a 0.85% aqueoussodium chloride solution to obtain 3.5 g of wet bacterial cells.

From the bacterial cell, a chromosomal DNA (hereinafter, referred to aschromosomal DNA (A)) was obtained by using QIAprep Genomic-tip System(manufactured by Qiagen).

Example 1 Obtaining of Invented DNA, and Analysis Thereof

(1) Preparation of Invented Protein

Into 10 ml of a 20 mM potassium phosphate buffer (pH 7.0) to which PMSF(phenylmethylsulfonyl fluoride) has been added so as to be 1 mM wassuspended about 7.5 g of the wet bacterial cells of the Stenotrophomonassp. SC-1 strain (FERM-BP 10785) prepared under the same condition asthat of Reference Example, and this was ground with a multi beadsshocker (manufactured by Yasui Kikai), glass beads 0.1 mmΦ, 2500 rpm, 20minutes). After 38 mg of protamine sulfate was added to the resultingground product, this was centrifuged (8000 rpm, 4° C., 10 minutes), andthe supernatant was further filtered with a filter (0.45 μm) to obtainabout 8 ml of the supernatant. The same procedure was repeated to obtainabout 40 ml of the supernatant.

About 10 ml of the resulting supernatant was applied on an affinityinteraction chromatography column [HiTrap BlueHP (manufactured byAmersham Pharmacia Biotech)] [equilibrated with a 20 mM potassiumphosphate buffer (pH 7.0)], and the column was eluted using, as a mobilephase, a potassium phosphate buffer with sodium chloride dissolvedtherein (concentration gradient of sodium chloride concentration 0→1.0M) to obtain 2 ml of a fraction at a sodium chloride concentration of0.5 to 0.8 M as a fraction having reductase activity. The same procedurewas repeated to obtain about 10 ml of an active fraction.

The eluted fraction was concentrated using Amicon Ultra-4 (manufacturedby MILLIPORE), and the buffer was exchanged with a 20 mM Tris-HCl buffer(pH7.5). This was applied on an ion exchange chromatography column[HiTrap DEAE Sepharose FF (manufactured by Amersham Pharmacia Biotech)][equilibrated with a Tris-HCl buffer solution (20 mM, pH 7.5)], and thecolumn was eluted using, as a mobile phase, a Tris-HCl buffer solutionwith sodium chloride dissolved therein (concentration gradient of sodiumchloride concentration 0→0.5 M) to obtain 2 ml of an active fraction ata sodium chloride concentration of 0.1 to 0.2 M as a fraction havingreductase activity. The same procedure was repeated to obtain about 6 mlof an active fraction.

This eluted fraction was concentrated using Amicon Ultra-4 (manufacturedby MILLIPORE), and the buffer was exchanged with a 50 mM sodiumphosphate buffer (pH 7.0) containing 0.15 M sodium chloride. Thisconcentrated solution was passed through a gel filtration column[Superdex200 10/300GL (manufactured by Amersham Pharmacia Biotech)[mobile phase: 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 Msodium chloride] to obtain 1 ml of an eluted fraction at a molecularweight of about 35000 dalton (hereinafter, referred to as activefraction (A)) as a fraction having reductase activity.

Regarding the fraction obtained by the chromatography and so on,reductase activity was measured by the following procedure.

Three (3) mg of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 0.2 mlof the eluted fraction obtained by the chromatography and so on, 9 mg ofNADPH, 0.15 ml of butyl acetate, and 1.5 ml of a 100 mM phosphate buffer(pH 7.0) were mixed, and the mixture was stirred at 30° C. for 18 hours.Thereafter, 2 ml of ethyl acetate was added to the reaction solution,and this was centrifuged to obtain an organic layer. The organic layerwas subjected to content analysis measurement by liquid chromatographyunder the following condition. The reductase activity was determinedfrom the residual amount of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, andthe production amount of(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.

(Content Analysis Condition)

Column: SUMICHIRAL ODS A-212

Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, Bsolution acetonitrile solution containing 0.1% trifluoroacetic acid

Time (min) A solution (%):B solution (%) 0 80:20 20 10:90 30  1:99 30.180:20Flow rate: 0.5 ml/minColumn temperature: 40° C.Detection: 290 nm

(2) Analysis of Amino Acid Sequence of Partial Peptide Derived fromInvented Protein

The active fraction (A) obtained by the above procedure was subjected toSDS polyacrylamide gel electrophoresis according to the method describein Laemmli, U. K., Nature, (1970) 227, 680. The gel afterelectrophoresis was stained with a Coomassie brilliant blue G250staining solution (manufactured by BIO-RAD), and a stained part of thegel was cut out. The gel was washed, and treated with trypsin, andpeptides were extracted from the gel. The extracted peptides werefractionated by HPLC (column: TSK gel ODS-80Ts, 2.0 mm×250 mm(manufactured by Tosoh), mobile phase: A solution (0.1% aqueoustrifluoroacetic acid), B solution (90% aqueous acetonitrile solutioncontaining 0.09% trifluoroactic acid), concentration gradient ofA/B=100/0→0/100). Two fractions from the obtained respective fractionswere subjected to an amino acid sequence analysis by protein sequencer(Procise 494HT Protein Sequencing System). The determined amino acidsequence is shown in SEQ ID No: 3 or SEQ ID No: 4.

(3) Analysis of Partial Nucleotide Sequence Derived from Invented DNA(I)

Based on the amino acid sequence shown in SEQ ID NO: 3, anoligonucleotide primer comprising the nucleotide sequence shown in SEQID NO: 5 was synthesized. Separately, based on the amino acid sequenceshown in SEQ ID NO: 4, an oligonucleotide primer comprising thenucleotide sequence shown in SEQ ID NO: 6 was synthesized.

Using the oligonucleotide primers each having the nucleotide sequencesshown in SEQ ID NO: 5 and SEQ ID NO: 6, and employing the chromosomalDNA (A) as a template, PCR was performed under the following reactionsolution composition and the reaction condition (using Expand HighFidelity PLUS PCR System manufactured by Roche Diagnostics).

[Reaction Solution Composition]

Chromosomal DNA (A) solution 2 μl dNTP (each 2.5 mM-mix) 1 μl Primer (50pmol/μl) each 0.4 μl 5 × buffer (with MgCl) 10 μl enz. expandHiFi (5U/μl) 0.5 μl Superpure water 35.7 μl

[Reaction Condition]

A container containing a reaction solution of the above composition wasset in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C.for 2 minutes, followed by 10 times of a cycle of 94° C. (10seconds)-65° C. (30 seconds)-72° C. (90 seconds) and, then, 20 times ofa cycle of 94° C. (10 seconds)-65° C. (30° C. seconds)-72° C. (1minute+5 second/cycle), and further, the container was incubated at 72°C. for 7 minutes.

Thereafter, an aliquot of the PCR reaction solution was taken and wassubjected to agarose gel electrophoresis resulting in detecting a bandof a DNA fragment of about 600 bp.

The DNA fragment of about 600 bp was ligated to the ready-made “PCRProduct insertion site” of a pCR2.1-TOPO vector (using TOPO TA cloningKit, manufactured by Invitrogen), and E. coli TOP10F′ was transformedwith the resulting ligation solution.

Thirty (30) μl of a 4% aqueous solution of 5-bromo-4-chloro-3-indolylβ-D-galactoside (hereinafter, referred to as X-gal) and 30 μl of 0.1 MIPTG were spread on an LB (1% Bacto-trypsin, 0.5% Bacto-yeast extract,1% sodium chloride) agar medium containing 50 μg/ml of ampicillin, andthe resulting transformants were inoculated thereon, followed byculturing. One white colony among formed colonies was taken, and thiscolony was inoculated in a sterilized LB medium (2 ml) containing 50μg/ml of ampicillin, and shaking-cultured (30° C., 24 hours) in a tube.A plasmid was taken out from the cultured bacterium cells using QIAprepSpin Miniprep Kit (manufactured by Qiagen).

A nucleotide sequence of the DNA fragment having been inserted into theresulting plasmid was analyzed, and the nucleotide sequence shown in SEQID NO: 7 was determined.

The analysis of the nucleotide sequence of the DNA fragment inserted inthe plasmid was performed by conducting a sequencing reaction using DyeTerminator Cycle sequencing FS ready Reaction Kit (manufactured byPerkin Elmer) and employing each plasmid as a template, and analyzingthe nucleotide sequence of the resulting DNA with a DNA Sequencer 373A(manufactured by Perkin Elmer).

(4) Analysis of Partial Sequence Derived from Invented DNA (II)

Based on the nucleotide sequence shown in SEQ ID NO: 7, anoligonucleotide primer comprising the nucleotide sequence shown in SEQID NO: 8, and an oligonucleotide primer comprising the nucleotidesequence shown in SEQ ID NO: 9 were synthesized. Separately, based onthe nucleotide sequence shown in SEQ ID NO: 7, an oligonucleotide primercomprising the nucleotide sequence shown in SEQ ID NO: 10, and anoligonucleotide primer comprising the nucleotide sequence shown in SEQID NO: 11 were synthesized.

Using the oligonucleotide primers comprising the nucleotide sequencesshown in SEQ ID NO: 8 and SEQ ID NO: 9 respectively, PCR was performedunder the following reaction solution composition and the reactioncondition using as a template a DNA obtained by treatment of thechromosomal DNA (A) with a restriction enzyme EcoRI followed byself-ligation with a 14 DNA ligase. In addition, using theoligonucleotide primers comprising the nucleotide sequences shown in SEQID NO: 10 and SEQ ID NO: 11 respectively, PCR was performed under thefollowing reaction solution composition and the reaction condition(using Expand High Fidelity PLUS PCR System manufactured by RocheDiagnostics) using as a template a DNA obtained by treatment of thechromosomal DNA (A) with a restriction enzyme EcoRV followed byself-ligation with a T4 DNA ligase.

[Reaction Solution Composition]

Chromosomal DNA (A) treatment solution 2 μl dNTP (each 2.5 mM-mix) 1 μlPrimer (50 pmol/μl) each 0.4 μl 5 × buffer (with MgCl) 10 μl enz.expandHiFi (5 U/μl) 0.5 μl Superpure water 35.7 μl

[Reaction Condition]

A container containing a reaction solution of the above composition wasset in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C.for 2 minutes, followed by 10 times of a cycle of 94° C. (10seconds)-65° C. (30 seconds)-72° C. (90 seconds) and, then, 20 times ofa cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5seconds/cycle), and further, the container was incubated at 72° C. for 7minutes.

Thereafter, an aliquot of the PCR reaction solution was taken, andsubjected to agarose gel electrophoresis. A band of a DNA fragment ofabout 600 bp was detected as a result of PCR performed usingoligonucleotide primers comprising the nucleotide sequences shown in SEQID NO: 8 and SEQ ID NO: 9. In addition, a band of a DNA fragment ofabout 1300 bp was detected as a result of PCR performed usingoligonucleotide primers comprising the nucleotide sequences shown in SEQID NO: 10 and SEQ ID NO: 11.

The DNA fragment of about 600 bp or about 1300 bp was ligated to theready-made “PCR Product insertion site” of apCR2.1-TOPO vector (usingTOPO™TA cloning Kit, manufactured by Invitrogen), and E. coli TOP10F′was transformed with the ligation solution.

Thirty (30) μl of an X-gal 4% aqueous solution and 30 μl of 0.1 M IPTGwere spread on an LB agar medium containing 50 μg/ml of ampicillin, andthe resulting transformants were inoculated thereon, followed byculturing. One white colony among formed colonies was taken, and thiscolony was inoculated in a sterilized LB medium (2 ml) containing 50μg/ml of ampicillin, and shaking-cultured (30° C., 24 hours) in a tube.A plasmid was taken out from each cultured bacterium cell using QIAprepSpin Miniprep Kit (manufactured by Qiagen).

A nucleotide sequence of the DNA fragment having been inserted in theresulting plasmid was analyzed. Based on the resulting nucleotidesequence, was determined the nucleotide sequence (SEQ ID NO: 2) encodingan amino acid sequence of a protein of Stenotrophomonas sp. SC-1 strain(FERM-BP 10785) having the ability to asymmetrically reduce2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide topreferentially produce(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.Further, based on SEQ ID NO: 2, the amino acid sequence (SEQ ID NO: 1)of the protein was determined.

When SEQ ID NO: 1 was compared with SEQ ID NO: 3 or SEQ ID NO: 4, it wasfound that the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4is identical with a part of the amino acid sequence shown in SEQ ID NO:1.

The analysis of the nucleotide sequence of the DNA fragment having beeninserted in the plasmid was performed by conducting a sequencingreaction using Dye Terminator Cycle sequencing FS ready Reaction Kit(manufactured by Perkin Elmer) and employing each plasmid as a template,and analyzing the nucleotide sequence of the resulting DNA with a DNAsequencer 373A (manufactured by Perkin Elmer).

Example 2 Example of Production of Invented Transformant and ReducingReaction (I)

(1) Preparation of Invented Vector

Based on the nucleotide sequence shown in SEQ ID NO: 2, anoligonucleotide primer comprising the nucleotide sequence shown in SEQID NO: 12, and an oligonucleotide primer comprising the nucleotidesequence shown in SEQ ID NO: 13 were synthesized.

Using the oligonucleotide primer comprising the nucleotide sequenceshown in SEQ ID NO: 12, and the oligonucleotide primer shown in SEQ IDNO: 13 as primers, and employing the chromosomal DNA (A) as a template,PCR was performed under the following reaction solution composition andthe reaction condition (using Expand High Fidelity PLUS PCR Systemmanufactured by Roche Diagnostics).

[Reaction Solution Composition]

Chromosomal DNA (A) solution 2 μl dNTP (each 2.5 mM-mix) 1 μl Primer (50pmol/μl) each 0.4 μl 5 × buffer (with MgCl) 10 μl enz. expandHiFi (5U/μl) 0.5 μl Superpure water 35.7 μl

A container containing a reaction solution of the above composition wasset in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C.for 2 minutes, followed by 10 times of a cycle of 94° C. (10seconds)-65° C. (30 seconds)-72° C. (90 seconds), and then, 20 times ofa cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5seconds/cycle), and further, the container was incubated at 72° C. for 7minutes.

Thereafter, an aliquot of the PCR reaction solution was taken andsubjected to agarose gel electrophoresis resulting in detection of aband of a DNA fragment of about 1000 bp.

Two kinds of restriction enzymes (NcoI and XbaI) were added to theremaining PCR reaction solution to double-digest the DNA fragment ofabout 1000 bp, and then, the enzyme-digested DNA fragment was purified.

Separately, a plasmid vector pTrc99A (manufactured by Pharmacia) wasdouble-digested with two kinds of restriction enzymes (NcoI and XbaI),and the enzyme-digested DNA fragment was purified.

These enzyme-digested DNA fragments were mixed, and ligated with a T4DNA ligase, and E. coli DH5α was transformed with the resulting ligationsolution.

The resulting transformants were cultured on an LB agar mediumcontaining 50 μg/ml of amplicillin, and 8 colonies were randomlyselected from the grown colonies. Each of the selected colonies wasinoculated in a sterilized LB medium (2 ml) containing 50 μg/ml ofamplicillin, and shaking-cultured (37° C., 17 hours) in a tube. Aplasmid was taken out from each cultured bacterial cell using QIAprepSpin Miniprep Kit (manufactured by Qiagen). An aliquot of each plasmidtaken out was double-digested with two kinds of restriction enzymes ofNcoI and XbaI, and then subjected to electrophoresis to confirm that theDNA fragment of about 1000 bp was inserted in each of the six plasmidstaken out (hereinafter, this plasmid is referred to as plasmid pTrcRs).

(2) Example of Preparation of Invented Transformant and ReducingReaction

E. coli HB101 was transformed using the plasmid pTrcRs. The resultingtransformant was inoculated in a sterilized LB medium (9 ml) containing0.2 mM of IPTG and 50 μg/ml of ampicillin, and shaking-cultured (37° C.,15 hours). The resulting culture was centrifuged to obtain 0.12 g of wetbacterial cells. The resulting wet bacterial cells were mixed with 1.5mg of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide,6.9 mg of NADPH, 1.5 ml of a 100 mM phosphate buffer (pH 7.0), 2.7 mg ofglucose, and 0.075 ml of butyl acetate, followed by stirring at 30° C.for 23 hours. Thereafter, 2 ml of ethyl acetate was added to thereaction solution, and this was centrifuged to obtain an organic layer.The organic layer was subjected to content analysis by liquidchromatography under the following condition, and it was found that2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide wasproduced at 97.9% relative to the amount of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide used inthe reaction. The optical purity of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide inthe organic layer was measured under the following condition. Theoptical purity of (R) body was found to be 100% e.e.

(Content Analysis Condition)

Column: SUMICHIRAL ODS A-212

Mobile phase: A solution 0.1% aqueous trifluoroactic acid solution, Bsolution acetonitrile solution containing 0.1% trifluoroactic acid

Time (min) A solution (%):B solution (%) 0 80:20 20 10:90 30  1:99 30.180:20Flow rate: 0.5 ml/minColumn temperature: 40° C.Detection: 290 nm

(Optical Purity Measuring Condition)

Column: CHIRALCEL OD-H

Mobile phase: hexane:2-propanol=9:1

Analysis time: 50 minutes

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Example 3 Example of Production of Invented Transformant and ReducingReaction (II)

(1) Arrangement for Preparing a Gene Comprising a Nucleotide SequenceEncoding an Amino Acid Sequence of a Protein Having Ability to ConvertOxidized β-Nicotineamide Adenine Dinucleotide into Reduced Form

Bacillus megaterium IFO12108 strain was cultured in 100 ml of asterilized LB medium to obtain 0.4 g of bacterial cells.

From the bacterial cells, a chromosomal DNA (hereinafter, referred to aschromosomal DNA (B)) was purified using Qiagen Genomic Tip (manufacturedby Qiagen) according to the method described in a manual attachedthereto.

(2) Preparation of a Gene Comprising a Nucleotide Sequence Encoding anAmino Acid Sequence of a Protein Having Ability to Convert Oxidizedβ-Nicotineamide Adenine Dinucleotide into Reduced Form

Based on the sequence of glucose dehydrogenase derived from Bacillusmegaterium IWG3 described in The Journal of Biological Chemistry Vol.264, No. 11, 6381-6385 (1989), an oligonucleotide primer comprising thenucleotide sequence shown in SEQ ID NO: 14, and an oligonucleotideprimer comprising the nucleotide sequence shown in SEQ ID NO: 15 weresynthesized.

Using the oligonucleotide primer comprising the nucleotide sequenceshown in SEQ ID NO: 14 and the oligonucleotide primer comprising thenucleotide sequence shown in SEQ ID NO: 15 as primers, and employing thechromosomal DNA (B) as a template, PCR was performed under the followingreaction solution composition and the reaction condition (using ExpandHigh Fidelity PCR System manufactured by Roche Diagnostics).

[Reaction Solution Composition]

Chromosomal DNA (B) stock solution 1 μl dNTP (each 2.5 mM-mix) 0.4 μlPrimer (20 pmol/μl) each 0.75 μl 10 × buffer (with MgCl) 5 μl enz.expandHiFi (3.5 × 10³ U/ml) 0.375 μl Superpure water 41.725 μl

[PCR Reaction Condition]

A container containing a reaction solution of the above composition wasset in PERKIN ELMER-GeneAmp PCR System2400, and was heated at 97° C. for2 minutes, followed by 10 times of a cycle of 97° C. (15 seconds) −55°C. (30 seconds) −72° C. (1.5 minutes), and then, 20 times of a cycle of97° C. (15 seconds)-55° C. (30 seconds)-72° C. (1 minute+5seconds/cycle), and further, the container was incubated at 72° C. for 7minutes.

Thereafter, an aliquot of the PCR reaction solution was taken, andsubjected to agarose gel electrophoresis resulting in detection of aband of a DNA fragment of about 950 bp.

The DNA fragment of about 950 bp obtained by PCR was ligated with theready-made “PCR Product insertion site” of pCR2.1-TOPO vector using theresulting PCR reaction solution and TOPO TA cloning Kit Ver. Emanufacturedby Invitrogen, and E. coli DH5α was transformed with theligation solution.

Thirty (30) μg of an X-gal 4% aqueous solution and 30 μl of 0.1M IPTGwere spread on an LB agar medium containing 50 μg/ml of ampicillin, andthe resulting transformants were inoculated thereon, followed byculturing. One white colony among formed colonies was taken, and thiscolony was inoculated in a sterilized LB medium (2 ml) containing 50μg/ml of ampicillin, and this was shaking-cultured (30° C., 24 hours) ina tube. Then, a plasmid was taken out from the cultured bacterial cellsusing QIAprep Spin Miniprep Kit (manufactured by Qiagen). An aliquot ofthe plasmid taken out was digested with the restriction enzyme (EcoRI),and it was subjected to electrophoresis, whereby it was confirmed thatthe DNA fragment of about 950 bp was inserted in the plasmid(hereinafter, this plasmid is referred to as plasmid pSDGDH12).

A nucleotide sequence of the DNA fragment having been inserted in thePlasmid pSDGDH12 was analyzed. The result is shown in SEQ ID No: 16.

The analysis of the nucleotide sequence of the DNA fragment having beeninserted in the plasmid was performed by conducting a sequencingreaction using Dye Terminator Cycle sequencing FS ready Reaction Kit(manufactured by Perkin Elmer), and employing the Plasmid pSDGDH12 as atemplate, and analyzing the nucleotide sequence of the resulting DNAwith DNA Sequencer 373A (manufactured by Perkin Elmer).

Then, based on the nucleotide sequence shown in SEQ ID NO: 16,oligonucleotide primers comprising the nucleotide sequences shown in SEQID NO: 17 and SEQ ID NO: 18 were synthesized.

PCR was performed using the oligonucleotide primes having the nucleotidesequences shown in SEQ ID NO: 17 and SEQ ID NO 18, and employing thechromosomal DNA (B) under the following reaction solution compositionand the reaction condition (using Expand High Fidelity PCR Systemmanufactured by Roche Diagnostics).

[Reaction Solution Composition]

Chromosomal DNA (B) stock solution 1 μl dNTP (each 2.5 mM-mix) 0.4 μlPrimer (20 pmol/μl) each 0.75 μl 10 × buffer (with MgCl) 5 μl enz.expandHiFi (3.5 × 10³ U/ml) 0.375 μl Superpure water 41.725 μl

[PCR Reaction Condition]

A container containing a reaction solution of the above composition wasset in PERKIN ELMER-GeneAmp PCR System2400, and was heated at 97° C. for2 minutes, followed by 10 times of a cycle of 97° C. (15 seconds)-55° C.(30 seconds)-72° C. (1.5 minutes), and then, 20 times of a cycle of 97°C. (15 seconds)-55° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle),and then, the container was incubated at 72° C. for 7 minutes.

Thereafter, an aliquot of the PCR reaction solution was taken, andsubjected to agarose gel electrophoresis resulting in detection of aband of a DNA fragment of about 800 bp.

Two kinds of restriction enzymes (NcoI and BamHI) were added to theremaining PCR reaction solution to double-digest the DNA fragment ofabout 800 bp, and then, the enzyme-digested DNA fragment was purified.

Separately, the plasmid vector pTrc99A (manufactured by Pharmacia) wasdouble-digested with two kinds of restriction enzymes (NcoI and BamHI),and the enzyme-digested DNA fragment was purified.

These enzyme-digested DNA fragments were mixed, and ligated with a T4DNA ligase, and E. coli DH5α was transformed with the resulting ligationsolution.

The resulting transformants were cultured on an LB agar mediumcontaining 50 μg/ml of ampicillin, and 10 colonies were randomlyselected from the grown colonies. Each of the selected colonies wasinoculated in a sterilized LB medium (2 ml) containing 50 μg/ml ofampicillin, and this was shaking-cultured (37° C., 17 hours) in a tube.A plasmid was taken out from each cultured bacterial cell using QIAprepSpin Miniprep Kit (manufactured by Qiagen). An aliquote of each plasmidtaken out was double-digested with two kinds of restriction enzymes ofNcoI and BamHI, and then subjected to electrophoresis to confirm thatthe DNA fragment of about 800 bp was inserted in each of four plasmidstaken out (hereinafter, this plasmid is referred to as Plasmid pTrcGDH).

(3) Preparation of Invented Vector

Using as primers an oligonucleotide primer comprising the nucleotidesequence shown in SEQ ID NO: 19 and an oligonucleotide primer shown inSEQ ID NO: 12, and employing the plasmid pTrcRs as a template, PCR wasperformed under the following reaction solution composition and thereaction condition (using Expand High Fidelity PCR System manufacturedby Roche Diagnostics).

[Reaction Solution Composition]

Plasmid pTrcRs 2 μl dNTP (each 2.5 mM-mix) 1 μl Primer (50 pmol/μl) each0.4 μl 5 × buffer (with MgCl) 10 μl Expand High Fidelity PLUS Taqpolymerase 0.5 μl (2.5 U) Superpure water 35.7 μl

[Reaction Condition]

A container containing a reaction solution of the above composition wasset in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C.for 2 minutes, followed by 10 times of a cycle of 94° C. (10seconds)-65° C. (30 seconds)-72° C. (90 seconds), and then, 20 times ofa cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5seconds/cycle), and further, the container was incubated at 72° C. for 7minutes.

Thereafter, an aliquote of the PCR reaction solution was taken, andsubjected to agarose gel electrophoresis resulting in detection of aband of a DNA fragment of about 1000 bp.

Two kinds of restriction enzymes (BamH and XbaI) were added to theremaining PCR reaction solution to double-digest the DNA fragment ofabout 1000 bp, and then, the enzyme-digested DNA fragment was purified.

Separately, the plasmid pTrcGDH was double-digested with two kinds ofrestriction enzymes (BamHI and XbaI), and the enzyme-digested DNAfragment was purified.

These enzyme-digested DNA fragments were ligated with a T4 DNA ligase,and E. coli DH5α was transformed with the ligation solution. Theresulting transformants were cultured on an LB agar medium containing 50μg/ml of ampicillin, and 6 colonies were randomly selected from growncolonies. Each of the selected colonies was inoculated in a sterilizedLB medium (2 ml) containing 50 μg/ml of ampicillin, and shaking-cultured(30° C., 7 hours) in a tube. A plasmid was taken out from each culturedbacterial cell using QIAprep Spin Miniprep Kit (manufactured by Qiagen).An aliquote of each plasmid taken out was double-digested with two kindsof restriction enzymes of BamHI and XbaI, and then subjected toelectrophoresis to confirm that the DNA fragment of about 1000 bp ofinterest was inserted in all of the plasmid taken out (hereinafter, thisplasmid is referred to as plasmid pTrcGSRs).

(4) Example of Preparation of Invented Transformant and ReducingReaction

Using the plasmid pTrcGSRs, E. coli HB101 was transformed. The resultingtransformant was inoculated in a sterilized LB medium (5 ml) containing0.2 mM of IPTG and 50 μg/ml of ampicillin, and shaking-cultured (30° C.,24 hours). The resulting culture was centrifuged to obtain about 0.1 gof wet bacterial cells. The resulting wet bacterial cells were mixedwith 3 mg of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 1.8 mgof NADP⁺, 1.5 ml of 100 mM phosphate buffer (pH 7.0), 2.8 mg of glucose,and 0.15 ml of tetrahydrofuran, followed by stirring at 30° C. for 24hours. Thereafter, 2 ml of ethyl acetate was added to the reactionsolution, and this was centrifuged to obtain an organic layer. Theorganic layer was subjected to content analysis by liquid chromatographyunder the following condition, and it was found that2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide isproduced at 96.8% relative to the amount of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide used inthe reaction. The optical purity of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide inthe organic layer was measured under the following condition. Theoptical purity of (R) body was found to be 100% e.e.

(Content Analysis Condition)

Column: SUMICHIRAL ODS A-212

Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, Bsolution acetonitrile solution containing 0.1% trifluoroacetic acid

Time (min) A solution (%):B solution (%) 0 80:20 20 10:90 30  1:99 30.180:20Flow rate: 0.5 ml/minColumn temperature: 40° C.Detection: 290 nm

(Optical Purity Measuring Condition)

Column: CHIRALCEL OD-H

Mobile phase: hexane:2-propanol=9:1

Analysis time: 50 minutes

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Example 4 Example of Reducing Reaction by Invented Transformant (III)

Using the plasmid pTrcRs, E. coli HB101 was transformed. The resultingtransformant was inculcated in a sterilized LB medium (5 ml) containing0.1 mM of IPTG and 50 μg/ml of ampicillin, and shaking-cultured (37° C.,15 hours). The resulting culture was centrifuged to obtain about 0.1 gof wet bacterial cells. The resulting wet bacterial cells were mixedwith 3 mg of methyl2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-oxoacetate, 13.5 mg of NADPH,1.5 ml of 100 mM phosphate buffer (pH 7.0), and 0.15 ml of butylacetate, followed by stirring at 30° C. for 24 hours. Thereafter, 2 mlof ethyl acetate was added to the reaction solution, and this wascentrifuged to obtain an organic layer. The organic layer was subjectedto content analysis by liquid chromatography under the followingcondition, and it was found that methyl2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-hydroxyacetate is produced at28.8% relative to the amount of methyl2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-oxoacetate used in thereaction. The optical purity of methyl2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-hydroxyacetate in the organiclayer was measured under the following condition. The optical purity of(R) body was found to be 100% e.e.

(Content Analysis Condition)

Column: SUMICHIRAL ODS A-212

Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, Bsolution acetonitrile solution containing 0.1% trifluoroacetic acid

Time (min) A solution (%):B solution (%) 0 80:20 20 10:90 30  1:99 30.180:20Flow rate: 0.5 ml/minColumn temperature: 40° C.Detection: 290 nm

(Optical Purity Measuring Condition)

Column: CHIRALCEL OD-H

Mobile phase: hexane:2-propanol=9:1

Analysis time: 50 minutes

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Example 5 Various Natures of Invented Protein

(1) Preparation of Invented Protein

Using the plasmid pTrcRs, E. coli HB101 was transformed. The resultingtransformant was inoculated in a sterilized LB medium (100 ml×8)containing 0.1 mM of IPTG and 50 μg/ml of ampicillin, and wasshaking-cultured (37° C., 13.5 hours). The resulting culture wascentrifuged to obtain about 5.4 g of wet bacterial cells. The wetbacterial cells (about 5.4 g) were suspended in 20 ml of 20 mM potassiumphosphate buffer (pH 7.0), and ground with a multibeads shocker(manufactured by Yasui Kikai, glass beads 0.1 mmΦ, 2500 rpm, 20minutes). The resulting ground product was centrifuged (8000 rpm, 4° C.,10 minutes), and the supernatant was further filtered with a filter(0.45 μm) to obtain about 18 ml of the supernatant. The resultingsupernatant was concentrated using Amicon Ultra-4 (manufactured byMILLIPORE) to obtain about 4 ml of the concentrated supernatant.

About 4 ml of the resulting concentrated supernatant was applied on anaffinity interaction chromatography column [HiTrap BlueHP (manufacturedby Amersham Pharmacia Biotech)] [equilibrated with a 20 mM potassiumphosphate buffer (pH 7.0)], and the column was eluted using, as a mobilephase, a potassium phosphate buffer with sodium chloride dissolvedtherein (concentration gradient of sodium chloride concentration 0→1.2M) to obtain about 6 ml of a fraction at a sodium chloride concentrationof 0.01 to 0.34 M as a fraction having reductase activity.

The eluted fraction was concentrated using Amicon Ultra-4 (manufacturedby MILLIPORE), and the buffer was exchanged with a 20 mM Tris-HCl buffer(pH 7.5). This was applied on an ion exchange chromatography column[HiTrap DEAE Sepharose FF (manufactured by Amersham Pharmacia Biotech)][equilibrated with a Tris-HCl buffer solution (20 mM, pH 7.5)], and thecolumn was eluted using, as a mobile phase, a Tris-HCl buffer solutionwith sodium chloride dissolved therein (concentration gradient of sodiumchloride concentration 0→0.5 M) to obtain 2 ml of an active fraction ata sodium chloride concentration of 0.1 to 0.2 M as a fraction havingreductase activity.

The eluted fraction was concentrated using Amicon Ultra-4 (manufacturedby MILLIPORE), and the buffer was exchanged with a 50 mM sodiumphosphate buffer (pH 7.0) containing 0.15 M sodium chloride. Theconcentrated solution was passed through a gel filtration column[Superdex200 10/300GL (manufactured by Amersham Pharmacia Biotech)][mobile phase: 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 Msodium chloride] to obtain 1 ml of an eluted fraction at a molecularweight of about 32200 dalton (hereinafter, referred to as activefragment (C)) as a fraction having reductase activity.

Regarding the fraction obtained by the chromatography and so on,reductase activity was measured by the following procedure.

Three (3) mg of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 0.2 mlof the eluted fraction obtained by chromatography and so on, 9 mg ofNADPH, 0.15 ml of butyl acetate, and 1.5 ml of a 100 mM phosphate buffer(pH 7.0) were mixed, and the mixture was stirred at 30° C. for 18 hours.Thereafter, 2 ml of ethyl acetate was added to the reaction solution,and this was centrifuged to obtain an organic layer. The organic layerwas subjected to content analysis measurement by liquid chromatographyunder the following condition. The reductase activity was determinedfrom the residual amount of2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, andthe production amount of(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.

(Content Analysis Condition)

Column: SUMICHIRAL ODS A-212

Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, Bsolution acetonitrile solution containing 0.1% trifluoroacetic acid

Time (min) A solution (%):B solution (%) 0 80:20 20 10:90 30  1:99 30.180:20Flow rate: 0.5 ml/minColumn temperature: 40° C.Detection: 290 nm

(2) Enzyme Chemical Nature of Invented Protein

An enzyme chemical nature of the invented protein was investigated usingthe active fragment (C).

(1) Action

Using NADPH as a coenzyme, the invented protein was acted on2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide toreduce to(R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamidehaving an optical purity of 100% e.e.

(2) Optimal pH

In order to investigate an acting optimal pH, the reduction activity ofethyl benzoylformate in a pH 5.5 to 10 was measured. As a buffer, a 100mM phosphate buffer (pH 5.5 to 8), a 100 mM Tris-Hcl buffer (pH 7 to 9),and a 100 mM Tris-glycine buffer (pH 9 to 10) were used. The reductionactivity on ethyl benzoylformate was obtained by dissolving a substrateethyl benzoylformate so as to be a final concentration of 10 mM, and acoenzyme NADPH so as to be a final concentration of 0.5 mM in variousbuffer solutions, adding the purified invented protein (active fragment(C)), performing a reaction at 30° C. for 1 minute, and calculating theactivity from a rate of reduction in absorbance of the reaction solutionat a wavelength of 340 nm. In the present reaction condition, theactivity of oxidizing 1 μmol of NADPH to NADP in one minute was definedas 1 unit. As a result, an optimal pH was 7.0.

(3) Substrate Specificity

In a 100 mM phosphate buffer solution (pH 7), a carbonyl compound as asubstrate was dissolved so as to be a final concentration of 1 mM, and acoenzyme NADPH was dissolved so as to be a final concentration of 0.5mM. An appropriate amount of the purified invented protein (activefragment (C)) was added thereto, followed by a reaction at 30° C. for 5minutes. From a rate of reduction in absorbance of the reaction solutionat a wavelength of 340 nm, the reduction activity on each carbonylcompound was calculated, and the activity was expressed as a relativevalue when activity on ethyl benzoylformate was made to be 100%, and theresults were shown in Table 1.

TABLE 1 Substrate Relative activity (%) Eethyl benzoylformate 100 Ethylpyruvate 33.7 Ethyl 4-phenyl2-oxobutyrate 1.5 Methyl4-bromo-3-oxobutyrate 3.9 Ethyl 4-chloro-3-oxobutyrate 0.53-Benzoylpropionic acid 3.3 Propionaldehyde 8.6 o-Chlorophenacylchloride 1.0

Example 6 Example of Reducing Reaction by Invented Transformant (IV) (1)Synthesis of ethyl 2-(2-methyl-phenyl)-2-oxoacetate

To a mixture of 50 g of tatrahydrofuran and 7.3 g of magnesium was added4.4 g of 2-bromotoluene to activate the magnesium. A temperature wasraised to 40° C., a solution of 45 g of 2-bromotoluene intetrahydrofuran (130 g) was added dropwise until the disappearance of2-bromotoluene was confirmed. Thereafter, the mixture was cooled to roomtemperature to prepare a Grignard reagent.

A solution of 83 g of diethyl oxalate in toluene (170 g) was cooled to−40° C. or lower. The Grignard reagent prepared as described above wasadded dropwise at −40° C. or lower, and this was retained at −40° C. for2 hours. To the reaction mixture was added 234 g of 10% sulfuric acid,and an organic layer was recovered. The resulting organic layer waswashed with 134 g of water, and dried with magnesium sulfate. Afterconcentration with an evaporator, distillation under high vacuum, andsilica gel column purification afforded 33.1 g of ethyl2-(methyl-phenyl)-2-oxoacetate.

¹H-NMR (300 MHz, CDCl₃): δppm: 1.41 (t, J=7.2 Hz, 3H), 2.61 (s, 3H),4.39-4.47 (m, 2H), 7.26-7.70 (m, 4H)

(2) Synthesis of 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide

The ethyl 2-(2-methyl-phenyl)-2-oxoacetate of 18.1 g obtained in (1), 72g of toluene, and 36 g of methanol were mixed, 23.2 g of a 40% aqueousmethylamine solution was added while retaining at 25° C., and themixture was retained at 25° C. for 1 hour. Thereafter, 36.2 g of waterwas added, and an organic layer was recovered. The layer was washed with143 g of 5% hydrochloric acid, 360 g of an aqueous saturated sodiumbicarbonate solution, and 36 g of water, respectively, and dried withmagnesium sulfate. After concentration with an evaporator, silica gelcolumn purification afforded 11.4 g of2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide.

¹H-NMR (300 MHz, CDCl₃): δppm: 2.48 (s, 3H), 2.96 (d, J=5.13 Hz, 3H),7.1 (6s, 1H), 7.25-7.93 (m, 4H)

(3) Asymmetrical reduction of2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide Using Invented Transformant

Using the plasmid pTrcGSRs, E. coli HB101 was transformed. The resultingtransformant was inoculated in a sterilized LB medium (100 ml)containing 0.1 mM of IPTG and 50 μg/ml of ampicillin, and wasshaking-cultured (37° C., 15 hours). The resulting culture wascentrifuged to obtain 0.5 g of wet bacterial cells. To a reaction tubewere added 30 mg of 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide, 0.5 gof the wet bacterial cells, 30 mg of NADP⁺, 45 mg of glucose, and 5 mlof a 100 mM phosphate buffer solution (pH 7), and these were reacted ata stirring rate of 1000 rpm at 30° C. for 24 hours. After the completionof the reaction, 5 ml of ethyl acetate was added to the reactionsolution, and this was centrifuged (1000×g, 5 minutes) to recover anorganic layer. The organic layer was distilled off to obtain 22.5 mg ofoily 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide. ¹H-NMR analysisresult of the resulting 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamideis shown below.

¹H-NMR (300 MHz, CDCl₃) δ2.37 (S, 3H), 2.79 (d, J=4.96 Hz, 3H), 3.87(1H), 5.15 (S, 1H), 6.21 (S, 1H), 7.15-7.26 (m, 4H)

To the resulting 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide wasadded hexane containing 10% 2-propanol, this was subjected to opticalpurity analysis by liquid chromatography under the following condition,and the optical purity was found to be 100% e.e. (elution time: 22.5minutes).

Racemic 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide was synthesizedby reducing 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide with NaBH₄ inmethanol.

(Optical Isomer Analysis Condition)

Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)

Mobile phase: hexane/2-propanol=90/10

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Elusion time

2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide: 16.0 minutes

2-(2-methyl-phenyl)-N-methyl-2-hydroxyacetamide: 18.5 minutes, 22.4minutes

Example 7 Example of Reducing Reaction by Invented Transformant (V)

A reaction was performed according to the same manner as in Example 6(3) except that ethyl2-(2-methyl-phenyl)-2-oxoacetate was used as asubstrate. As a result, 26.5 mg of oily ethyl2-(2-methyl-phenyl)-2-hydroxy-acetate was obtained. ¹H-NMR analysisresult of the resulting ethyl2-(2-methyl-phenyl)-2-hydroxy-acetate isshown below.

¹H-NMR (300 MHz, CDCl₃) δ1.21 (t, J=7.2 Hz, 3H), 2.43 (S, 3H), 3.56 (d,J=7.7 Hz, 1H), 4.13-4.29 (m, 2H), 5.35 (S, 1H), 7.15-7.30 (m, 4H)

To the resulting ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate was addedhexane containing 10% 2-propanol, this was subjected to optical purityanalysis by liquid chromatography under the following condition, and anoptical purity was found to be 99% e.e. (elution time: 13.3.minutes).

Racemic ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate was synthesized byreducing ethyl2-(2-methyl-phenyl)-2-oxoacetate with NaBH₄ in methanol.

(Optical Isomer Analysis Condition)

Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)

Mobile phase: hexane/2-propanol=90/10

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Elusion time

ethyl 2-(2-methyl-phenyl)-2-oxoacetate: 8.9 minutes

ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate: 11.6 minutes, 13.3 minutes

Example 8 Example of Reducing Reaction by Invented Transformant (VI) (1)Synthesis of ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate

A temperature of 59.8 g of a 28% solution of sodium methoxide inmethanol was raised to 60° C., a mixture solution of 70.4 g ofo-bromobenzyl bromide and 70.4 g of methanol was added dropwise, andthis was retained at 60° C. for 2 hours. After the mixture was cooled toroom temperature, 211 g of toluene and 211 g of water were addedthereto, the mixture was stirred, and layers were separated, and anorganic layer was recovered. An aqueous layer was extracted with 211 gof toluene three times, the recovered organic layer and 211 g of waterwere added, and this was washed, and concentrated to obtain 55.3 g of1-methoxymethyl-2-bromobenzene.

To a mixture of 46.4 g of tetrahydrofuran and 5.4 g of magnesium wasadded 4.6 g of 1-methoxymethyl-2-bromobenzene to activate the magnesium.A temperature was raised to 40° C., a solution of 41.7 g of1-methoxymethyl-2-bromobenzene in tetrahydrofuran (139 g) was addeddropwise until the disappearance of 1-methoxymethyl-2-bromobenzene wasconfirmed. Thereafter, the mixture was cooled to room temperature toprepare a Grignard agent.

A solution of 64.4 g of diethyl oxalate in toluene (177 g) was cooled to−40° C. or lower. The Grignard agent prepared as described above wasadded dropwise at −40° C. or lower, and this was retained at −40° C. for4 hours. To the reaction mixture was added 211 g of 10% sulfuric acid,and an organic layer was recovered. The resulting organic layer waswashed with 133 g of water, and dried with magnesium sulfate. Afterconcentration with an evaporator, silica gel column purification anddistillation under the high pressure afforded 38.5 g of ethyl2-(2-methoxymethyl-phenyl)-2-oxoacetate.

¹H-NMR (300 MHz, CDCl₃): δppm: 1.41 (t, J=7.08 Hz, 3H), 3.44 (s, 3H),4.41 (q, 2H), 4.75 (s, 2H), 7.55-7.69 (m, 4H)

(2) Synthesis of 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide

28.3 g of the ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate obtained in(1), 109 g of toluene, and 54.4 g of methanol were mixed, 28.6 g of a40% aqueous methylamine solution was added while retaining at 25° C.,and the mixture was retained at 25° C. for 2 hours. Thereafter, 54.4 gof water was added, 80 g of toluene were further added, this was allowedto stand still, and an organic layer was recovered. The organic layerwas washed with 178 g of 5% hydrochloric acid, 54.4 g of an aqueoussaturated sodium bicarbonate solution, and 54.4 g of water,respectively, and concentrated with an evaporator to obtain 12.0 g of2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide.

¹H-NMR (300 MHz, CDCl₃): δppm: 2.95 (d, J=5.14 Hz, 3H), 3.28 (s, 3H),4.66 (s, 2H), 7.04 (s, 1H), 7.36-7.72 (m, 4H)

(3) Asymmetric Reduction of2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide Using InventedTransformant

A reaction was performed according to the same manner as in Example 6(3)except that 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide was usedas a substrate. As a result, 19.5 mg of oily2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide was obtained.¹H-NMR analysis result of the resulting2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide is shown below.

¹H-NMR (300 MHz, CDCl₃) δ2.77 (d, J=4.85 Hz, 3H), 3.47 (S, 3H), 4.38 (d,J=10.6 Hz, 1H), 4.73 (d, J=10.6 Hz, 1H), 4.81 (S, 1H), 5.23 (S, 1H),7.16 (S, 1H), 7.26-7.43 (m, 4H)

To the resulting 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxyacetamidewas added hexane containing 10% 2-propanol, this was subjected tooptical purity analysis by liquid chromatography under the followingcondition, and the optical purity was found to be 100% e.e. (elutiontime: 20.3 minutes).

Racemic 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxyacetamide wassynthesized by reducing2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide with NaBH₄ inmethanol.

(Optical Isomer Analysis Condition)

Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)

Mobile phase: hexane/2-propanol=90/10

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Elusion time

2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide: 18.5 minutes

2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide: 20.4 minutes,21.0 minutes

Example 9 Example of Reducing Reaction by Invented Transformant (VII)

A reaction was performed according to the same manner as in Example 6(3)except that ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate was used as asubstrate. As a result, 27.6 mg of oily ethyl2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate was obtained.

¹H-NMR analysis result of the resulting ethyl2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate is shown below.

¹H-NMR (300 MHz, CDCl₃) δ1.21 (t, J=7.09 Hz, 3H), 3.39 (S, 3H),4.16-4.26 (m, 2H), 4.59 (d, J=6.36 Hz, 2H), 5.39 (S, 1H), 7.31-7.37 (m,4H)

To the resulting ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate wasadded hexane containing 10% 2-propanol, this was subjected to opticalpurity analysis by liquid chromatography under the condition, and theoptical purity was found to be 100% e.e. (elution time: 13.6 minutes).

Racemic ethyl2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate wassynthesized by reducing ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetatewith NaBH₄ in methanol.

(Optical Isomer Analysis Condition)

Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)

Mobile phase: hexane/2-propanol=90/10

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 230 nm

Elusion time

ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate: 9.3 minutes

ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate: 12.8 minutes, 13.6minutes

INDUSTRIAL APPLICABILITY

According to the present invention, optically-active mandelic acidcompounds can be easily obtained.

1. An isolated DNA comprising any nucleotide sequence of the followinga) to d): a) a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO:1; b) a nucleotide sequence of a DNA: i) wherein the DNA hasat least 90% sequence homology with a DNA comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:1, and ii)wherein the nucleotide sequence encodes an amino acid sequence of aprotein having the ability to asymmetrically reduce an ortho-substitutedphenylglyoxalic acid compound to produce corresponding optically activeortho-substituted mandelic acid; c) a nucleotide sequence of a DNA: i)wherein the DNA hybridizes under a stringent condition including washingfor 30 minutes at 65° C. in 0.1×SSC with a DNA comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:1, and ii)wherein the nucleotide sequence encodes an amino acid sequence of aprotein having the ability to asymmetrically reduce an ortho-substitutedphenylglyoxalic acid compound to produce corresponding optically-activeortho-substituted mandelic acid compound; and d) the nucleotide sequenceof SEQ ID NO:2.
 2. A DNA in which a promoter functional in a host celland the DNA according to claim 1 are operably linked.
 3. A recombinantvector comprising the DNA according to claim
 1. 4. A transformant inwhich the DNA of claim 2 has been introduced into a host cell.
 5. Thetransformant according to claim 4, wherein the host cell is amicroorganism.
 6. The transformant according to claim 4, wherein thehost cell is Escherichia coli.
 7. A transformant comprising the DNA ofclaim
 1. 8. A process for producing a transformant comprising a step ofintroducing the recombinant vector of claim 3 into a host cell.
 9. Anisolated protein comprising any amino acid sequence of the following a)to e); a) the amino acid sequence of SEQ ID NO:1; b) an amino acidsequence: i) which is encoded by a nucleotide sequence of a DNA havingat least 90% sequence homology with a DNA comprising the nucleotidesequence of SEQ ID NO:2, and ii) which is an amino acid sequence of aprotein having the ability to asymmetrically reduce an ortho-substitutedphenylglyoxalic acid compound to produce a correspondingoptically-active ortho-substituted mandelic acid compound; c) an aminoacid sequence: i) which is encoded by a nucleotide sequence of a DNAwhich hybridizes under a stringent condition including washing for 30minutes at 65° C. in 0.1×SSC with a DNA comprising the nucleotidesequence of SEQ ID NO:2, and ii) which is an amino acid sequence of aprotein having the ability to asymmetrically reduce an ortho-substitutedphenylglyoxalic acid compound to produce a correspondingoptically-active ortho-substituted mandelic acid compound; d) an aminoacid sequence: i) in which one amino acid is deleted, substituted oradded in the amino acid sequence of SEQ ID NO:1, and ii) which is anamino acid sequence of a protein having the ability to asymmetricallyreduce an ortho-substituted phenylglyoxalic acid compound to produce acorresponding optically-active ortho-substituted mandelic acid compound;and e) an amino acid sequence: i) which has at least 90% sequencehomology with the amino acid sequence of SEQ ID NO:1, and ii) which isan amino acid sequence of a protein having the ability to asymmetricallyreduce an ortho-substituted phenylglyoxalic acid compound to produce acorresponding optically-active ortho-substituted mandelic acid compound.10. A recombinant vector comprising the DNA of claim 1 and a DNAcomprising a nucleotide sequence encoding an amino acid sequence of aprotein having the ability to convert oxidized β-nicotineamide adeninedinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphateinto a reduced form.
 11. The recombinant vector according to claim 10,wherein the protein having the ability to convert oxidizedβ-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adeninedinucleotide phosphate into a reduced form is glucose dehydrogenase. 12.The recombinant vector according to claim 11, wherein the protein havingglucose dehydrogenase activity is glucose dehydrogenase derived fromBacillus megaterium.
 13. A transformant, in which the recombinant vectorof claim 10 has been introduced into a host cell.
 14. The transformantaccording to claim 13, wherein the host cell is a microorganism.
 15. Thetransformant according to claim 13, wherein the host cell is Escherichiacoli.
 16. A transformant comprising the DNA of claim 1 and a DNAcomprising a nucleotide sequence encoding an amino acid sequence of aprotein having the ability to convert oxidized β-nicotineamide adeninedinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphateinto a reduced form.
 17. A process for producing an optically-activealcohol compound comprising reacting the protein of claim 9, or thetransformant of any of claims 4, 7, 13 and 16, or a treated productthereof with a prochiral carbonyl compound.
 18. The process according toclaim 17, wherein the prochiral carbonyl compound is an amide or estercompound of the ortho-substituted phenylglyoxalic acid, and theoptically-active alcohol compound is an amide or ester compound of theoptically-active ortho-substituted mandelic acid.
 19. The processaccording to claim 18, wherein the amide or ester compound of theortho-substituted phenylglyoxalic acid is a compound represented by theformula (1):

(wherein R₁ represents an optionally substituted amino group, or anoptionally substituted alkoxy group, and R₂ represents an optionallysubstituted C1-8 alkyl group), and the amide or ester compound of theoptically-active ortho-substituted active mandelic acid is anoptically-active compound represented by the formula (2):

(wherein R₁ and R₂ are as defined above, and a carbon atom with a *symbol is an asymmetric carbon atom).
 20. Stenotrophomonas sp. SC-1(FERM BP-10785).